Enzyme-Substrate-Cofactor Dynamical Networks Revealed by High-Resolution Field Cycling Relaxometry

Area of Science:

• Biochemistry
• Enzymology
• Structural Biology

Background:

• Enzyme catalysis relies on dynamic interactions between enzymes, substrates, and cofactors.
• The role of protein dynamics in enzyme function is well-studied, but ligand dynamics are less understood.
• Guanosine-5 ext-monophosphate reductase (GMPR) provides a model system with catalytically competent complexes mimicking reaction intermediates.

Purpose of the Study:

• To investigate the dynamics of enzyme-bound substrates and cofactors in GMPR using sub-tesla high-resolution field cycling 31P NMR relaxometry.
• To identify structural features contributing to distinct dynamic signatures in hydride transfer and deamination complexes.
• To elucidate the role of specific amino acid residues in modulating cofactor conformation and ammonia binding.

Main Methods:

• Utilized sub-tesla high-resolution field cycling 31P NMR relaxometry to probe ligand dynamics.
• Employed site-directed mutagenesis to perturb cofactor conformation and ammonia binding in GMPR.
• Conducted exchange experiments to assess ammonia/ammonium affinity for different enzyme complexes.

Main Results:

• Asp129 is integral to dynamic networks for both hydride transfer and deamination.
• Lys77 modulates substrate and cofactor mobility in a reaction-specific manner.
• Thr105 and Tyr318 form a deamination-specific network involving the GMP 2 ext-OH, with minimal impact on hydride transfer dynamics.
• Ammonia/ammonium exhibits high affinity for the deamination complex and low affinity for the hydride transfer complex, potentially gating cofactor conformational changes.

Conclusions:

• Enzyme, substrates, and cofactors are interconnected in intricate, reaction-specific dynamic networks.
• Distal regions of substrates and cofactors are critical components of these dynamic networks.
• High-resolution field cycling NMR relaxometry is a powerful tool for investigating ligand dynamics in enzymatic systems.